Benzimidazole Resistance In Nematodes Research Articles

PCR-RFLP screening of polymorphisms associated with benzimidazole resistance in Necator americanus and Ascaris lumbricoides from different geographical regions in Brazil.

Ascaris lumbricoides and Necator americanus are soil-transmitted parasites with global geographic distribution, and they represent some of the most common and neglected infections in the world. Periodic treatment with mass drug administration (MDA) in endemic areas is the recommended action put forth by the World Health Organization. However, MDA can cause the selection of subpopulations that possess the genetic ability to overcome the mechanism of drug action. In fact, beta-tubulin gene mutations (codons 167, 198 and 200) are correlated with benzimidazole resistance in nematodes of veterinary importance. It is possible that these SNPs also have strong correlation with treatment resistance in the human geohelminths A. lumbricoides, Trichuris trichiura and hookworms. Here, we aimed to investigate the presence of some of these canonical molecular markers associated with parasite resistance to benzimidazole in N. americanus and A. lumbricoides collected from six Brazilian states. Nested-PCR and PCR-RFLP were used to detect mutations at codons 167 and 198 in 601 individual eggs of A. lumbricoides collected from 62 human stool samples; however, no mutations were found. Codons 198 and 200 were tested in 552 N. americanus eggs collected from 48 patients using the same methodology, which presented a relative frequency of 1.4% and 1.1%, respectively. The presence of these SNPs in N. americanus eggs is an important finding, indicating that with high benzimidazole drug pressure there is potential for benzimidazole resistance to be selected in this hookworm. However, at these low frequencies it does not indicate that there is at present any benzimidazole resistance problem. This is the first systematic study performed in South America, and the study yielded a landscape of the genetic variants in the beta-tubulin gene and anthelmintic resistance to soil-transmitted parasites detected by a simple, rapid and affordable genotyping assay of individual eggs.

Variability of the egg hatch assay to survey benzimidazole resistance in nematodes of small ruminants under field conditions

The egg hatch assay (EHA) is one of the main in vitro methods for detection of benzimidazole resistance in nematode parasites of small ruminants. However, although the EHA has been standardised at the laboratory level, the diagnostic performance of this method has not been fully characterised for field screenings. In the present work, monthly variation of benzimidazole resistance estimated by EHA was surveyed over two years in three sheep flocks and in one goat and an additional sheep flock sharing the same pastures. Resistance was measured by calculating both the effective dose of thiabendazole (TBZ) that inhibited hatching of ≥50% of parasite eggs (ED50) and the proportion (Pdd) of eggs hatching at a discriminating dose of 0.1μg/ml TBZ. Pdd exhibited higher variability than ED50, in agreement with the higher sensitivity of Pdd to changes in resistance levels. Both resistance parameters, however, were highly correlated, and their variation was similarly related to the same factors. Resistance levels differed among sheep flocks, and the resistance level of the goat flock was higher than that measured for the sheep flock sharing the same pasture. Moreover, monthly variation of resistance in goats did not mirror that recorded in sheep. Resistance levels varied seasonally, with the highest values recorded in the spring, and they were inversely related to the number of days that samples were stored under anaerobic conditions. In addition, they were directly associated with the relative abundance of Teladorsagia spp. but inversely related to the relative abundance of Haemonchus spp. After controlling for the effects of these identified factors for variation, inter-monthly sampling variation due to unknown factors was the main source of variability, accounting for more than 60–70% of variance for both resistance parameters and yielding absolute estimation errors higher than 0.06 for ED50 or 0.2 for Pdd when resistance was estimated from a single sampling. Optimum sample size, estimated from variance components, suggested that at least 4–5 samplings would be needed to halve this absolute error, whereas additional samplings would slightly increase precision but at the cost of substantially increasing sampling effort. More research is needed to identify the main factors involved in this inter-sampling variation to standardise the implementation of EHA under field conditions.

Alternation of anthelmintic treatments: A molecular evaluation for benzimidazole resistance in nematodes

The evolution of benzimidazoles (BZ) resistance in Teladorsagia circumcincta was investigated in a controlled trial with lambs, submitted to different treatment regimens. Four paddocks were seeded with a T. circumcincta strain constituted by 25% of BZ-resistant nematodes. Ten permanent lambs were allocated to each paddock, from April to November in order to renew the contamination of pasture. Monthly, three tracer lambs were allocated in each paddock. BZ-resistant nematode frequency was determined (PCR diagnosis). The faecal egg count reduction test (permanent lambs) and the number of nematodes in lambs were also determined (permanent and tracer lambs). Four different regimens of treatments were performed: control, levamisole (a non-BZ drug), fenbendazole (a BZ drug), and an alternation of levamisole and fenbendazole every second treatment. The same protocol was repeated on two consecutive grazing seasons, increasing the number of treatments (3 in first year and 5 in second year). The proportions of BZ-resistant nematodes did not change during all the study in both the control and the levamisole paddocks, supporting an equal global fitness of BZ-resistant and susceptible nematodes. Thus, no reversion of BZ resistance is to be expected. In the alternated drug group and in the BZ treated group, BZ-resistant nematodes increased from 25% to 47% and to 78%, respectively. BZ resistance increased proportionally to the selective pressure (number of BZ treatments). The drug alternation is not a good solution to delay importantly the evolution of resistance when more than 25% of nematodes are BZ-resistant. This study is the first evaluation of BZ-resistance evolution (using individual genotyping) in controlled conditions. It showed that when a monogenic anthelmintic resistance is established at 25% in a sexually reproducing nematode population, it seems to be impossible to prevent its increase even when using limited number of BZ treatments.

Multiple resistance to anthelmintics in sheep nematodes and comparison of methods used for their detection

Intensification of animal production systems has led to an increased reliance on effective anthelmintics to control parasitic worms. However, the excessive and continued use of these “wormers” can lead to high selection pressures and have resulted in increased reports of emerging nematode populations exhibiting resistance to all of the main anthelmintic classes. Faecal egg count reduction tests (FECRT) were conducted on six farms in England and Wales according to standardised guidelines produced by the World Association for the Advancement of Parasitology. Selected farms were identified from a network of 40 study farms participating in a study investigating implementation of Sustained Control of Parasites in Sheep (SCOPS) principles. Resistance to the larval development test (LDT) to either benzimidazole- or imidazothiazole-group anthelmintics had been previously detected on these farms. In this study, resistance was indicated as present by LDT and/or FECRT to one or more groups of anthelmintics on a number of the study farms. Comparisons were made between results obtained by the two tests on the six farms. Benzimidazole resistance was identified by FECRT on five farms; imidazothiazole resistance on four farms and macrocyclic lactones resistance on five farms. The LDT identified the presence of benzimidazole resistance on all six farms, and imidazothiazole resistance on five farms. Generally, there was good agreement between the two tests in identifying both benzimidazole and imidazothiazole resistance. On one farm, the LDT identified the presence of benzimidazole-resistant nematodes, not detected by FECRT and on two farms the presence of imidazothiazole-resistant nematodes not detected by FECRT. On two farms “triple” resistance (i.e. resistance to all three groups of anthelmintics) was identified by FECRT and on one farm moxidectin resistance was suspected based on an early return to egg laying at 28 days post-treatment. Resistance was present in one or more genera, but most commonly in Teladorsagia on all six farms.

Genetic analysis of a relationship between macrocyclic lactone and benzimidazole anthelmintic selection on Haemonchus contortus

We have found that in a laboratory strain of the nematode parasite of ruminants, Haemonchus contortus, repeated ivermectin treatment, in vivo in sheep, selected against TTC, coding for Phe, and for TAC, coding for Tyr, at codon 200 in beta-tubulin isotype 1. This 200Tyr single nucleotide polymorphism (SNP) has been associated with benzimidazole anthelmintic resistance. In this study, we investigated the alpha-tubulin and beta-tubulin sequences of 17 different field and laboratory strains/isolates of H. contortus with known treatment history and status for susceptibility or resistance to macrocyclic lactone (ML) or benzimidazole anthelmintics. DNA (genomic or cDNA) from 10 to 32 male parasites was sequenced for alpha-tubulin or beta-tubulin for each strain/isolate and the frequency of coding SNPs was compared between the different strains/isolates with known treatment history and drug resistance status. We have analyzed alpha-tubulin and beta-tubulin isotypes 1 and 2 genes from the different H. contortus strains/isolates and found that repeated ivermectin or moxidectin (both ML anthelmintics) use changes the frequency of beta-tubulin isotype 1 alleles and selects for the following SNPs: either from TTC (Phe) to TAC (Tyr) at codon 200 or codon 167, or from GCA (Ala) to GAA (Glu) at codon 198. 200Tyr and 167Tyr were associated with GTT (Val) or CTC/CTT (Leu) at codon 368, whereas 200Phe or 167Phe were associated with either ATT (Ile) or GTT (Val) at codon 368 in beta-tubulin isotype 1. The SNPs 200Tyr, 167Tyr in beta-tubulin isotype 1 have been described to confer benzimidazole resistance in nematodes and recently 198Ala has also been associated with benzimidazole resistance. Although MLs exert their antiparasitic effect through binding to ligand-gated chloride channels, benzimidazoles interact with beta-tubulin. These genetic analyses indicate that there is a correlation between exposure and/or resistance to MLs and an increase in the frequency of the beta-tubulin alleles containing codons, which are determinant for benzimidazole resistance. ML use may predispose parasitic nematodes to benzimidazole resistance. This has major implications for parasite control programs dependent on MLs/benzimidazole rotations and on the use of ML/benzimidazole combination therapy, such as for human lymphatic filariasis and nematode control in ruminant livestock.

P-glycoprotein selection in strains of Haemonchus contortus resistant to benzimidazoles

Anthelmintic resistance in parasitic nematodes of livestock is a chronic problem in many parts of the world. Benzimidazoles are effective, broad-spectrum anthelmintics that bind to and selectively depolymerise microtubules. Resistance to the benzimidazoles, however, developed quickly and is caused by genetic changes in genes encoding β-tubulins, subunits of microtubules. In Haemonchus contortus, resistance to avermectins has been correlated with genetic changes at a gene encoding a P-glycoprotein, a cell membrane transport protein that has a very high affinity for ivermectin. The substrate specificity of P-glycoprotein is very broad, and resistance to benzimidazoles can be modulated by lectins specific for P-glycoprotein. We investigated the possibility that genetic changes in P-glycoprotein might be correlated with benzimidazole resistance in nematodes. An analysis of restriction fragment length polymorphisms of a P-glycoprotein gene from a sensitive and a cambendazole-selected strain of H. contortus, derived from the sensitive strain, showed a significant difference in allele frequencies between strains. The frequency of one allele in particular increased substantially. The same allele was also found at a high frequency in an independently derived thiabendazole-selected field isolate. We present genetic evidence of selection at a P-glycoprotein locus during selection for benzimidzole resistance in H. contortus.

Phenotyping and genotyping of Haemonchus contortus isolates reveals a new putative candidate mutation for benzimidazole resistance in nematodes

In order to monitor and eventually control the spread of drug-resistant Haemonchus contortus, knowledge of the molecular mechanisms underlying resistance is essential. Here we phenotypically and genotypically characterize three multidrug-resistant H. contortus field isolates from Australia and South Africa. All were significantly less susceptible to thiabendazole than a sensitive reference strain in an in vitro egg-hatch assay. The sensitivity was further reduced in a surviving population after treatment of infected sheep with albendazole. The β-tubulin genes were amplified from genomic DNA of the H. contortus isolates, cloned, and sequenced. There was a high degree of sequence variation. The known mutation phenylalanine-200 to tyrosine (F200Y) occurred in 60% of the sequences from resistant isolates, but not in the sensitive reference. Interestingly, 90% of the β-tubulin sequences from resistant isolates lacking tyrosine-200 carried another mutation nearby, glutamate-198 to alanine (E198A). This mutation was not found in the sensitive isolate, nor in sequences from resistant isolates carrying the mutation F200Y. However, the mutation E198A is known from benomyl-resistant isolates of phytopathogenic fungi such as Monilinia fructicola. The finding that alanine-198 correlates with thiabendazole resistance in H. contortus isolates from South Africa and Australia suggests that also in nematodes, the mutation E198A plays a role in benzimidazole resistance. Alanine-198 alleles of β-tubulin can be detected by PCR-RFLP and we suggest to include this test in future surveys of H. contortus field populations.

A possible model of benzimidazole binding to β-tubulin disclosed by invoking an inter-domain movement

Although it is well established that benzimidazole (BZMs) compounds exert their therapeutic effects through binding to helminth β-tubulin and thus disrupting microtubule-based processes in the parasites, the precise location of the benzimidazole-binding site on the β-tubulin molecule has yet to be determined. In the present study, we have used previous experimental data as cues to help identify this site. Firstly, benzimidazole resistance has been correlated with a phenylalanine-to-tyrosine substitution at position 200 of Haemonchus contortus β-tubulin isotype-I. Secondly, site-directed mutagenesis studies, using fungi, have shown that other residues in this region of the protein can influence the interaction of benzimidazoles with β-tubulin. However, the atomic structure of the αβ-tubulin dimer shows that residue 200 and the other implicated residues are buried within the protein. This poses the question: how might benzimidazoles interact with these apparently inaccessible residues? In the present study, we present a mechanism by which those residues generally believed to interact with benzimidazoles may become accessible to the drugs. Furthermore, by docking albendazole-sulphoxide into a modelled H. contortus β-tubulin molecule we offer a structural explanation for how the mutation conferring benzimidazole resistance in nematodes may act, as well as a possible explanation for the species-specificity of benzimidazole anthelmintics.

Evaluation of a larval development assay (DrenchRite ®) for the detection of anthelmintic resistance in cyathostomin nematodes of horses

A larval development assay (LDA, DrenchRite ®) was evaluated to determine the effectiveness of this method in detecting anthelmintic resistance in cyathostomin nematodes of horses. A total of 15 horse farms from Georgia and South Carolina (USA) and Population S ponies from the University of Kentucky (USA) were included in this study. Nematode eggs were extracted from pooled fecal samples and placed into the wells of a DrenchRite ® plate for testing against thiabendazole (TBZ), levamisole (LEV) and 2 ivermectin (IVM) analogs (IVM-1, IVM-2). After a 7-day incubation larvae in each well were counted and data were analyzed by logistic regression. Resistance status of each farm for different drugs was determined in a separate study using a fecal egg count reduction test. LDA were performed on the 15 farms once, however, the Population S cyathostomins were assayed on 3 separate occasions to estimate the consistency of results between assays. Mean TBZ LC 50 for oxibendazole resistant, suspected resistant and sensitive farms were 0.2015, 0.1625, and 0.1355 μM, respectively. For LEV, mean LC 50 for PYR resistant, suspected resistant and sensitive farms were 1.590, 1.8018 and 1.4219 μM, respectively. All 15 farms had worms susceptible to IVM; mean LC 50 for IVM-1 and for IVM-2 were 7.5727 and 87.9718 nM, respectively. A linear mixed model was fitted to the data to determine the relationship between LC 50 and LC 95 and resistance status for each farm. No meaningful relations were found. Consistency of assays varied between drugs, being best for TBZ and worst for LEV and IVM-1. All farms in this study had benzimidazole-resistant nematodes; therefore usefulness of DrenchRite ® for discriminating susceptibility versus resistance to this drug class could not be accurately assessed. Moreover, since all farms tested were sensitive to IVM and resistance to this drug class has not yet been reported in cyathostomins, it is not possible to assess accurately the usefulness of DrenchRite ® LDA for detecting IVM resistance at this time. Assay results for LEV suggest that LEV in a LDA does not yield data that is useful in estimating PYR efficacy in vivo. Based on results for PYR/LEV, the current high prevalence of benzimidazole resistance, no known cases of IVM resistance, and the sometimes extreme variation in results seen in many of the assays, DrenchRite ® LDA cannot be considered a useful tool for the diagnosis of resistance in cyathostomins of horses at present.

Dose-dependent activity of albendazole against benzimidazole-resistant nematodes in sheep: relationship between pharmacokinetics and efficacy

The relationship between the pharmacokinetic behaviour and the anthelmintic efficacy of albendazole (ABZ) against benzimidazole (BZD)-resistant nematodes was studied in sheep. A micronized ABZ suspension was orally administered at two different dose levels to sheep naturally infected with BZD-resistant gastrointestinal (GI) nematodes. The experimental animals were allocated into the following groups ( n=8): (a) untreated control; (b) orally treated with ABZ at 3.8 mg/kg b.w.; and (c) orally treated with ABZ at 7.5 mg/kg b.w. Plasma samples were obtained serially over 72 h post-treatment from both treated groups and analysed by HPLC to measure the concentrations of ABZ and its sulphoxide (ABZSO) and sulphone (ABZSO 2) metabolites. Faecal egg counts were performed prior to treatment and at the necropsy day. All experimental animals were sacrificed 10 days after treatment to perform GI worm counts. While ABZ parent drug was not recovered in the bloodstream, ABZSO and ABZSO 2 were the molecules found in plasma. ABZSO was the metabolite measured at the highest concentrations in the bloodstream for up to 36 (treatment at 3.8 mg/kg) or 60 h (treatment at 7.5 mg/kg) post-administration. There was a proportional relationship between the administered ABZ dose and the measured plasma concentrations of both ABZ metabolites. Over a 100% increment on the plasma AUC values for the anthelmintically active ABZSO metabolite was observed at the 7.5 mg/kg compared to the 3.8 mg/kg treatment. The low efficacy patterns (<24%) observed against the GI nematodes investigated indicate a high level of resistance to ABZ given at 3.8 mg/kg an efficacious therapeutic dose rate recommended in some countries. However, the higher and prolonged plasma drug concentration measured after the 7.5 mg/kg treatment resulted in an improved efficacy pattern (estimated by both faecal egg and adult worm counts) against most of the GI nematodes studied compared to that obtained at the lower dose rate. A direct relationship between drug pharmacokinetic behaviour and anthelmintic efficacy against BZD-resistant nematodes in sheep was shown in the current work, although individual variation precluded the observation of statistically significant differences in worm counts.

Prevalence of benzimidazole resistant nematodes in sheep flocks in Yucatan, Mexico

Thirty-eight sheep flocks, located in three municipalities in the Eastern Yucatan, Mexico, were surveyed for gastrointestinal nematodes resistant to benzimidazole (BZD) anthelmintics (AH). On each flock, 30 sheep were randomly distributed into two groups of 15 animals: albendazole group (5 mg/kg BW) and untreated control group. Animals were refrained from any food (either browsing/grazing or supplement) for a period of 16 h prior to treatment. Faecal egg counts (FEC) and larval cultures were performed 10 days after anthelmintic treatment. Percentage reduction and 95% confidence intervals were determined. Anthelmintic resistance (AR) was declared when the percentage reduction in FEC was <95% and the 95% confidence interval was <90%. AR was suspected when only one of the two criteria was met. The survey indicated that AR occurred in 15.8% ( n=6) (95% confidence interval=±11.6%) and was suspected in 23.7% of the farms ( n=9) (95% confidence interval=±13.3%). Post-treatment larval cultures indicated that Haemonchus was the only resistant genus. The questionnaire survey showed that most farmers (92%) considered their sheep a secondary activity to cattle production. The majority of farmers (97.4%) treat their animals according to visual appreciation of weight. However, most farmers (79%) treat their flocks at very low frequencies (<3 times per year). Drug rotation was performed every 12 months or more by 84.2% of farmers. Anthelmintics used were: macrociclic lactones (47.4%), BZD (39.5%), levamisol (10.5%) and 1 farmer used closantel.

Biological control of infective larvae of a gastro-intestinal nematode ( Teladorsagia circumcincta ) and a small lungworm ( Muellerius capillaris ) by Duddingtonia flagrans in goat faeces

The high prevalence of benzimidazole-resistant nematodes in French grazing dairy goat flocks led to a search for nematode-control schemes based on integrated approaches with non-chemical options, like vaccination, grazing management, or biological control using nematophagous fungi. The effect of the daily feeding of goats with spores of the nematophagous fungus Duddingtonia flagrans on third-stage larvae (L3) of Teladorsagia circumcincta was examined in faecal cultures. In addition, the effect of D. flagrans on the survival of first-stage larvae (L1) of Muellerius capillaris was tested. Twenty-two culled dairy goats previously raised in a zero-grazing system were twice infected at monthly intervals with 5,000 and then 7,500 T. circumcincta L3. Eight animals were infected with a benzimidazole-susceptible (BZs) strain while the remainder received a benzimidazole-resistant one (BZr). Six culled goats naturally infected with M. capillaris were purchased from private farms. All the goats were divided in two groups, one group receiving daily 5 x 10(5) chlamydospores of D. flagrans/kg body weight per goat for seven consecutive days in the food, the other group acting as control. For T. circumcincta-infected goats, individual egg counts and coprocultures (13 days, 25 degrees C) followed by L3 extraction with the Baermann method were performed. For M. capillaris-infected goats, extraction of L1 with the Baermann apparatus was individually performed on day 0 and after coprocultures on days 7, 10 and 14. Reductions in percentage development of T. circumcincta L3 in fungus groups compared with control groups ranged from 84% (BZs strain) to 90% (BZr strain). A decrease in M. capillaris L1 recovery was noted on days 7 and 10 (a reduction of 70% compared with day 0) and on day 14 (85%), but this pattern was similar in both groups, whether receiving the fungus or not. At the dosage of 5 x 10(5) spores/kg body weight, D. flagrans was highly effective in reducing the larval development of T. circumcincta in goats faeces. In contrast, the fungus did not reduce M. capillaris L1 survival in faeces in our conditions.

Efficacy of copper oxide needles for the control of nematode parasites in dairy goats.

The spread of benzimidazole-resistant nematodes in dairy goat farms is of a great concern as probably more than 70% of the flocks are involved. As there are very few other anthelmintic options during the lactating period, we have evaluated the efficacy of copper oxide needles (CON, Copinox, Bayer, UK) in both experimental and natural infections in goats. The curative effect of CON (2-4 g) on existing worm burdens was assessed in goats experimentally infected with Teladorsagia circumcincta, Haemonchus contortus and Trichostrongyus colubriformis, compared to controls. The preventive effect of CON (4 g) on worm establishment was monitored for 2 months in animals experimentally infected with H. contortus and for 3 months in naturally infected animals on a farm exhibiting predominant infections with T. circumcincta and Oesophagostomum venulosum. In both experimental and natural conditions, the efficacy of CON was nil against Teladorsagia, Trichostrongylus and Oesophagostomum infections. In contrast, the efficacy of CON against Haemonchus was clearly established in reducing the worm burden (75%) as well as in lowering the egg output (37-95%) in relation to the establishment of new infections over several weeks. Copper oxide needles may represent an alternative to conventional anthelmintics in the control of Haemonchus infection in some goat farms.

Economic evaluation of three anthelmintic strategies for lamb flocks affected by benzimidazole-resistant nematodes.

To compare the profitability of three anthelmintic strategies in growing lambs in flocks with nematodes resistant to benzimidazole anthelmintics. A partial-budgeting analysis was carried out by means of a stochastic simulation model, which allows inputs to be described as distributions rather than as fixed values, and hence permits variation between farms to be considered in the analysis. The results show that control of nematode parasites by use of an effective anthelmintic provides the highest net returns, yielding a margin over ineffectively treated lambs of A$114 per 100 lambs on average. Suppressive treatment based on the administration of two controlled-release capsules and monthly with moxidectin resulted in an average loss of A$131 per 100 lambs in comparison with animals treated with an ineffective anthelmintic. Analysis of the results from capsule-treated lambs did not take into account the unmeasured benefits associated with less contamination of pastures. Sensitivity analysis using a stochastic model indicates that apart from the effect of treatment on weight gain variation in carcase price greatly influences the profitability of all the parasite control programs examined. The results suggest that it is economically important for farmers to adjust their strategy in the presence of anthelmintic resistance. But as a result of uncertainty in the factors influencing economic return, the expected economic benefit is likely to vary substantially.

Prevalence of anthelmintic resistant nematodes in sheep and goats in Western France

Twenty-three sheep farms and 15 dairy goat farms, located in western France and randomly selected, were surveyed for gastrointestinal nematodes resistant to anthelmintics. On each of the 23 sheep farms, 60 lambs were randomly distributed into four groups of 15: untreated control group, fenbendazole (benzimidazole) group (5 mg/kg BW), ivermectin group (0.2 mg/kg BW) and levamisole hydrochloride group (6.8 mg/kg BW). On each of the 15 goat farms, 30 adult goats were allocated into two groups of 15: untreated control group and fenbendazole group (10 mg/kg BW). Faecal egg counts and larval cultures were done 10 to 14 days after anthelmintic treatment. Furthermore, egg hatch assays conducted with thiabendazole were performed on pooled faeces of fenbendazole treated goats, results being expressed as Logarithmic Concentration 50 (LC 50). Faecal egg count reductions (FECR) less than 95% are indicative of anthelmintic resistance and LC 50 values above 0.1 μg/ml suggest benzimidazole (BZ) resistance. On this basis, in the sheep survey, anthelmintic resistance occurred on 83% of farms for benzimidazole and on 50% of farms for levamisole. Post-treatment larval cultures indicated Teladorsagia/ Trichostrongylus and Cooperia were the predominant resistant genera. A negative relationship was found between FECR with fenbendazole and the average number of annual BZ treatments given to ewe-lambs ( P<0.01). In contrast, no case of ivermectin resistant nematodes was seen in the 23 sheep farms. In goat survey, virtually all the 15 surveyed farms showed the occurrence of benzimidazole resistant nematodes according to the results of both FECR test and egg hatch assay. In post-treatment coprocultures, Teladorsagia/ Trichostrongylus was the predominant larval type on seven farms, and Haemonchus on two farms, while on the remaining farms there was a well-balanced mixture of all the genera including Oesophagostomum/ Chabertia. Although no relationship could be established between drenching practices and results of in vivo and in vitro tests, the frequent and sometimes exclusive use of BZ compounds associated with a permanent underdosing of these drugs achieved by using sheep dose rates are probably the main causes of the very high frequency of BZ resistance in nematodes on goats. The consequences of anthelmintic resistance on sheep and goat parasitism management are discussed.

Anthelmintic resistance

Anthelmintic resistance is widespread in nematode parasites of sheep, goats and horses. Resistance is also developing in nematode parasites of cattle and has been detected in pig parasites. Benzimidazole, levamisole/morantel and ivermectin resistances occur in nematodes of sheep and goats and closantel resistance has been found in Haemonchus contortus. Anthelmintic resistance is likely to develop wherever anthelmintics are frequently used and be detected if it is investigated. Worm count or egg count reduction after treatment are useful for the detection of all types of anthelmintic resistances. More economical, faster and more sensitive in vitro assays for the detection of anthelmintic resistance have been developed. Some, such as the egg hatch assay are specific for a particular class of anthelmintic, whilst others such as larval development assays can be used with most anthelmintics. Improvements in our understanding of the biochemistry and molecular genetics of anthelmintic actions should lead to the development of more sensitive assays for the detection of anthelmintic resistance in individual nematodes. Levamisole/morantel resistance appears to be associated with alterations in cholinergic receptors in resistant nematodes. Ivermectin appears to act by binding to a glutamate receptor of a membrane chloride channel. This receptor has been expressed in vitro so that further studies of the interaction of ivermectin with this receptor and its possible alteration in ivermectin resistance will be feasible. Benzimidazole resistance in nematodes and fungi appears to be associated with an alteration in β-tubulin genes which reduces or abolishes the high affinity binding of benzimidazoles for tubulin in these organisms. This knowledge can be exploited for DNA probes for benzimidazole resistance/susceptibility in individual organisms.

Activity of morantel citrate against strains of benzimidazole-resistant nematodes of sheep in the United Kingdom

The activity of morantel citrate (5.94 mg/kg base) was determined in laboratory tests against field isolates of benzimidazole-resistant nematodes. Its efficacies against adult and seven-day-old worms were 100 per cent and 100 per cent for Cooperia curticei, 95.1 per cent and 69.8 per cent for Haemonchus contortus and 100 per cent and 82 per cent for Ostertagia circumcincta. Morantel citrate was 100 per cent effective against benzimidazole-susceptible Nematodirus battus and Trichostrongylus vitrinus, and it reduced faecal egg counts by 97.9 per cent in sheep infected naturally with benzimidazole-resistant H contortus and O circumcincta.

World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) methods for the detection of anthelmintic resistance in nematodes of veterinary importance

Methods have been described to assist in the detection of anthelmintic resistance in strongylid nematodes of ruminants, horses and pigs. Two tests are recommended, an in vivo test, the faecal egg count reduction test for use in infected animals, and an in vitro test, the egg hatch test for detection of benzimidazole resistance in nematodes that hatch shortly after embryonation. Anaerobic storage for submission of faecal samples from the field for use in the in vitro test is of value and the procedure is described. The tests should enable comparable data to be obtained in surveys in all parts of the world.

A survey of benzimidazole resistant nematodes in sheep in three countries of southern England

Faecal samples were collected from 209 randomly selected sheep farms in southern England, and the nematode eggs extracted were tested by an egg hatch assay and by a larval development test for their resistance to benzimidazoles. Benzimidazole resistance was found on 35 per cent of farms tested in East Sussex, 44 per cent in Oxfordshire and 61 per cent in West Sussex.

Benzimidazole resistant nematodes in sheep in southern England

Benzimidazole resistant nematodes in sheep in southern England